Malonate is relevant to the lung environment and induces genome-wide stress responses in Pseudomonas aeruginosa

Versatility in carbon source utilization is a major contributor to niche adaptation in Pseudomonas aeruginosa. Malonate is among the abundant carbon sources in the lung airways, yet it is understudied. Recently, we characterized how malonate impacts quorum sensing regulation, antibiotic resistance, and virulence factor production in P. aeruginosa. Herein, we show that malonate as a carbon source supports more robust growth in comparison to glycerol in several cystic fibrosis isolates of P. aeruginosa. Furthermore, we show phenotypic responses to malonate were conserved among clinical strains, i.e., formation of biomineralized biofilm-like aggregates, increased tolerance to kanamycin, and increased susceptibility to norfloxacin. Moreover, we explored transcriptional adaptations of P. aeruginosa UCBPP-PA14 (PA14) in response to malonate versus glycerol as a sole carbon source using transcriptomics. Malonate utilization activated glyoxylate and methylcitrate cycles and induced several stress responses, including oxidative, anaerobic, and metal stress responses associated with increases in intracellular aluminum and strontium. We identified several genes that were required for optimal growth of P. aeruginosa in malonate. Our findings reveal important remodeling of P. aeruginosa gene expression during its growth on malonate as a sole carbon source that is accompanied by several important phenotypic changes. These findings add to the accumulating literature highlighting the role of different carbon sources in the physiology of P. aeruginosa and its niche adaptation.


Introduction
Pseudomonas aeruginosa is an opportunistic pathogen that infects various hosts including humans [1].It can infect a wide range of body sites, e.g., lungs in cystic brosis patients, wounds and blood circulation in burn and trauma patients [2].Its versatile metabolism enables this bacterium to survive different environmental conditions by utilizing a multitude of carbon sources [3].This metabolic versatility is controlled by complex regulatory networks [4].The utilization of certain carbon sources is linked to antibiotic tolerance and altered virulence of P. aeruginosa [5][6][7].Furthermore, the activation of metabolic networks by speci c carbon sources involves a stress response depending on the environmental conditions [8,9].However, the link between carbon source utilization and stress response is not clear.
Glycerol is a byproduct of phosphatidylcholine, a major lung surfactant.Glycerol is recognized as a key carbon source for P. aeruginosa and has been included in Pseudomonas Isolation Agar (PIA), a traditional lab medium used for selective growth of the bacterium in which glycerol serves as a carbon source and enhances pyocyanin production.Yet, it is unknown if other available carbon sources in the lung may be advantageous for the growth of P. aeruginosa.One example is malonate, a naturally occurring organic acid, which is utilized by several microorganisms as a carbon source [6, [10][11][12][13].
Previous studies suggest the potential clinical importance of malonate.For example, malonate utilization genes are overexpressed in P. aeruginosa grown ex vivo in blood from trauma patients [6].Moreover, malonate increases the tolerance of P. aeruginosa to aminoglycoside antibiotics and in uences its quorum sensing circuit and virulence factors [5,6,14].These malonate-induced shifts in metabolism were also associated with the production of surface-free bio lm-like aggregates (also known as ocs) embedded in a biomineralized matrix [14].Similar bacterial aggregates have been associated with bio lms and lung infections [15,16].However, it is not known how carbon source utilization is involved in this phenotype.
Herein, we aim to focus on the clinical relevance of malonate if it can support the growth of cystic brosis (CF) isolates of P. aeruginosa as a sole carbon source.Furthermore, we study the transcriptional regulation occurring in P. aeruginosa during malonate utilization.Our ndings highlight the lifestyle modulation effect of carbon sources on P. aeruginosa and provide evidence of malonate-induced stress response and its associated phenotypes in this pathogen.

Results
Malonate is abundant in the human airways and promotes the growth of P. aeruginosa cystic brosis isolates The human airway is a complex environment with a multitude of metabolites that can serve as a carbon source for P. aeruginosa.Glycerol, a degradation product of the major lung surfactant phosphatidylcholine, has been recognized as a key carbon source for P. aeruginosa.Furthermore, glycerol has been included in Pseudomonas Isolation Agar (PIA), a traditional lab medium selective growth of P. aeruginosa, in which glycerol serves as a carbon source and enhances pyocyanin production.However, many metabolites are present in the lung and may serve as carbon sources for P. aeruginosa, e.g., succinate (Fig. 1A) [17].Unlike glycerol, malonate has not been well-studied, and whether it can support the growth of P. aeruginosa cystic brosis (CF) isolates is still unknown.
To this end, we used a library of 24 clinical isolates of P. aeruginosa (Table 1) to compare their growth in M9, a minimal medium, containing malonate (MM9) as a sole carbon source vs. glycerol as a control (GM9).While the majority (17 isolates) were able to grow in GM9 and MM9, ve isolates grew poorly, and two showed no growth (Fig. 1B).Only three isolates showed higher growth in GM9 vs. MM9 (Fig. 1C).Surprisingly, more than half of the clinical isolates showed more robust growth in MM9 rather than in GM9 (Fig. 1C).This was the same observation with P. aeruginosa strain UCBPP-PA14 (PA14), a commonly used lab strain, as well as other lab strains (Fig. 1D, Table 2).Conservation of malonate-associated phenotypes across clinical and lab strains Previously, we observed strong phenotypes for PA14 grown in MM9, i.e., aggregation and differential susceptibility to antibiotics [14].Therefore, we wanted to test if these phenotypes are conserved across clinical and lab strains.First, we examined if the bacterial culture of P. aeruginosa can form aggregates in MM9 versus GM9 media.We found that aggregation is a common outcome of malonate-utilization among the isolates (around two-thirds showed high aggregation index) (Fig. 2A).Additionally, all lab strains showed aggregation in MM9 (Fig. 2B).
Next, we wanted to examine if both clinical and lab strains exhibited increased tolerance to kanamycin and greater susceptibility to nor oxacin in MM9 compared to GM9 [18,19].We examined the impact of malonate and glycerol as carbon sources on antibiotic susceptibility using a broth microdilution assay for both kanamycin (up to 1200 µg/ml) and nor oxacin (up to 100 µg/ml compared to GM9 (Fig. 2C and Table 3).However, unlike control strains, CF MUC 13 and CF MUC 100 displayed heightened kanamycin tolerance in GM9 compared to MM9.Furthermore, several tested strains maintained consistent susceptibility or tolerance to kanamycin regardless of the carbon source.CF MUC 7 did not grow in MM9 but thrived in GM9.While we did observe growth for CF MUC7 in MM9 in shaking growth conditions as shown previously, we believe that a static growth set up could have resulted for this no growth phenotype.Interestingly, CF MUC 7 did not grow in GM9 when glacial acetic acid was used as a vehicle control.In addition, all strains demonstrated greater tolerance to nor oxacin in GM9 media than MM9 (Fig. 2C and Table 3).In summary, our results indicate that both lab and clinical P. aeruginosa strains tend to exhibit increased tolerance to kanamycin and heightened susceptibility to nor oxacin when malonate is utilized as a sole carbon source.Overall, these results suggest that while some phenotypes associated with malonate utilization are conserved across clinical and lab strains, others are strain speci c.Transcriptional activation of the glyoxylate and methylcitrate cycles by malonate Motivated by the phenotypes observed in the presence of malonate in comparison to glycerol, we aimed to decipher how malonate utilization alters P. aeruginosa at the transcriptional level.We examined the transcriptomic changes in PA14 when grown in MM9 vs. GM9.Transcriptomic data analysis was performed using Rockhopper 2 [20].Overall, 2,478 genes were differentially expressed (Benjamini-Hochberg procedure-adjusted P-value ≤ 0.05).In comparison to GM9, MM9 induced the expression of 1,370 genes and downregulated the expression of 1,108 genes.We validated our transcriptomic results, obtained from RNA-seq analysis, by examining the expression of a subset of genes of interest using qRT-PCR (Supplementary Fig. 1).
The gene expression data enabled us to establish which metabolic pathways were activated by the growth of PA14 on malonate as a sole carbon source.As expected, the growth of P. aeruginosa in minimal medium with only malonate supplemented as a sole carbon source upregulated the expression of the 10 genes involved in malonate utilization, i.e., regulation, uptake, and decarboxylation (Supplementary Fig. 2).The average expression ratio of these malonate utilization-related genes was 135 times higher in MM9 than in GM9.
Next, we identi ed 534 differentially expressed genes mapped to KEGG metabolic pathways, of which 70 mapped to carbon metabolism.Speci cally, over 50 genes were mapped to pathways involving tricarboxylic acid (TCA), glyoxylate, and methylcitrate cycles (Fig. 3).Upon utilization of malonate, acetyl-CoA is produced, which then feeds carbon metabolic circuits.Unlike the inhibited TCA cycle genes, the genes of glyoxylate and methylcitrate cycles were activated upon malonate utilization.These ndings provide insight into how malonate utilization regulates different carbon metabolic pathways which could be a crucial strategy for its adaptation in different niches.

Stress response is induced by malonate-utilization
Next, we investigated whether malonate utilization is linked to eliciting stress response in P. aeruginosa.Surprisingly, we identi ed over a hundred genes that are differentially expressed and linked to various stress responses.These can be broadly grouped as metal stress response (Table 4), oxidative stress response (Table 5), or carbon starvation/anaerobic stress response (Table 6), and a few general stress response genes that we grouped into a temperature, osmolarity, and pH stress response category (Table 7).

Metals and oxidative stress response
Our transcriptomic analysis revealed that malonate utilization relative to glycerol utilization induces genes that are associated with copper toxicity response in PA14 (Table 4) [21].Genes representing the copper resistance regulon include the operon consisting of the ve genes PA14_18810-18860 that were upregulated an average of 103.5 times in MM9 relative to GM9.Two copper-related genes, PA14_18800 and PA14_18070, were induced 7.2 and 46.8 times, respectively.These genes code for proteins with a copper chaperone CopZ domain.Three genes PA14_18760-18790 coding for a homolog of the resistance-nodulation-division (RND) e ux pump MexPQ-OpmE were also induced with an average of 15.6 times.This e ux pump was observed to be activated by copper [22].PA14_13170, a copA1/cueA homolog (a P-type ATPase), was induced 21.3 times.These copper resistance-regulated genes are known to be controlled by the transcriptional regulator CueR, and mutations in any of these genes result in a higher sensitivity to copper [21].Surprisingly, this gene's homolog (PA14_63170) was not differentially expressed (P-value = 0.2).Several other genes related to metal stress response were differentially regulated with malonate utilization.For example, the four genes coding for the RND e ux pump, MexGHI-OpmD, were upregulated with an average of 23.5 times.Other genes included those coding for cation-transporting P-type ATPases (Table 4).Lastly, PA14_65000 that codes for azurin was upregulated 3.7 times.We also observed that the genes involved in pyoverdine biosynthesis and uptake were downregulated in MM9, suggesting that the cell is shutting down its metal import systems (Table 4).The pvdD gene was downregulated 3 times, while the pyoverdine biosynthesis protein PvdE was downregulated 5 times.These results suggest that malonate utilization results in metal stress response due to either the accumulation of metals accompanied by malonate utilization or an alteration in the regulatory network controlling metals sensing independent of metal concentration.
Because oxidative stress is strongly linked to metal stress, we aimed next to investigate if oxidative stress-related genes were also differentially regulated by malonate utilization [23,24].Indeed, over a dozen genes involved in oxidative response were upregulated (Table 5).Catalase-coding genes (katA and katB) were upregulated 54.5 and 34.4 times, but katE was downregulated 3.1 times.PA14_61020 is a gene in the same operon as katB was found to be upregulated 11.1 times.Its gene product has an ankyrin repeat domain, which plays a role in signal transduction [25].PA14_21530 is another gene harboring ankyrin repeat domain in its protein product that was signi cantly upregulated 112.2 times.Interestingly, the expression of this gene is positively regulated by MvfR [26] and associated with oxidative stress adaptation [27].PA14_22320 is another gene that was signi cantly upregulated 184.6 times.It is possibly controlled by MvfR [26], and its expression is induced by hydroxyl radicals [28].PA14_03090 was another gene that was upregulated, and evidence suggests that it is required for hydrogen peroxide resistance [27].Three alkyl hydroperoxide reductase-coding genes (ahpB, ahpC, and ahpF) were upregulated with an average of 76.3 times-among them, ahpB differential expression was the highest, 171.1 times.Five peroxidase-coding genes were upregulated such as glutathione peroxidase, thioredoxin reductase 2, and cytochrome c551 peroxidase.These results are consistent with our previous nding where we observed an increase in the catalase activity of PA14 grown in MM9 [14].
Pyomelanin is a pigment produced by P. aeruginosa, and one of its main functions is to provide protection against oxidative stress [29].Our results suggest that malonate utilization is associated with its upregulation (Table 5).Genes PA14_52990 and PA14_53070, which are involved in the biosynthesis of homogentisate (the precursor of pyomelanin) from chorismate were upregulated.Moreover, genes (PA14_57830, PA14_57850, and PA14_57870) involved in the transport of homogentisate [30] and possibly the extracellular accumulation of pyomelanin were upregulated as well.Overall, these results suggest that malonate utilization in P. aeruginosa is accompanied by an oxidative stress response.

Carbon starvation and anaerobic stress response
Just as metal stress and oxidative stress are inseparably linked, carbon starvation and anaerobic stress response share many facets.Transcriptomic analysis of P. aeruginosa during malonate utilization reveals that P. aeruginosa is undergoing carbon starvation and anaerobic stress response (Table 6).The expression of rpoS, which encodes a sigma-factor known to be associated with carbon or oxygen limitation [31], was upregulated 3.5 times.The expression of psrA, an rpoS transcriptional regulator [32], was also upregulated 1.6 times.The aa 3 -type cytochrome c oxidase, encoded by the coxBA-PA14_01310-coIII gene cluster and induced by RpoS upon carbon limitation [3], was also found to be upregulated an average of 4.4 times.
We also identi ed a putative ABC transporter encoded by PA14_69090, PA14_69070, and PA14_69060 that was upregulated ~ 3 times.A previous study suggests that this ABC transporter is also required during oxygen limitation [32].The expression of the outer-membrane protein encoded by oprG, was upregulated 31 times.OprG is a speci c transporter of hydrophobic molecules and its expression is induced under anaerobic conditions [36].
Five genes encoding proteins with the universal stress protein family domains were upregulated (Table 6).These included uspO, uspK, uspN, uspL, and uspM with an average increase of 18.7 times.These genes play a role in response to different stressors-more importantly, anaerobic stress conditions or oxygen limitation [37].All genes of arcDABC operon, which is responsible for arginine fermentation, were upregulated.The expression of uspK, uspN and arcDABC was reported to be induced in bio lms or anaerobic environments [38].Two genes (acka and pta) involved in pyruvate fermentation and anaerobic metabolism [39] were also upregulated.Moreover, the cbb 3 -type cytochrome c oxidase, encoded by PA14_44340 and PA14_44350, was also upregulated.This system is induced under an oxygen limited environment [40].Lastly, another recently identi ed gene that is associated with survival under low oxygen conditions, mhr, was found to be upregulated almost 27 times [41].
The dnr gene (PA14_06870) encoding the transcriptional regulator Dnr was upregulated 9 times (Table 6).Dnr senses nitric oxide [42] and regulates the denitri cation gene [43].During the denitri cation process, nitrate is used as an alternative terminal electron acceptor and reduced to nitrogen [44].Our results showed that 20 genes that are part of the denitri cation operons (nar, nir, nap, nor, and nos) were induced.Our nding of increased expression of many nos, nor, nir, and dnr genes by cells suggests that P. aeruginosa is exposed to anaerobic conditions when grown in malonate as a sole carbon source.The fhp gene, and its transcriptional regulator fhpR, were upregulated as well.This avohemoglobin system is nitric oxide-responsive and impairs the dispersal response to nitric oxide [45].These results suggest that nitric oxide is produced during malonate utilization, thus eliciting an anaerobic (low oxygen) stress response.This was supported by the upregulation in the three genes constituting hcn operon that is responsible for the synthesis of hydrogen cyanide, which is induced under hypoxic environment [46].Moreover, the recently characterized small RNA, sicX, encoded by PA14_46160 was upregulated ~ 6 times.It is worth noting that sicX was found to be induced by low oxygen and is important in the transition between chronic and acute infection stages in P. aeruginosa [47].
Eight genes involved in the biosynthesis of pyocyanin were signi cantly upregulated (Table 6).For example, phzB 1 , one of the main biosynthetic operons phz 1 , was upregulated 109.5 times.But four genes of the redundant operon phz 2 were downregulated.Analysis of the differential regulation of these biosynthetic operons, though note-worthy, is beyond the scope of this study.This possible increase in pyocyanin ts with our hypothesis, as pyocyanin is important for P. aeruginosa to maintain redox homeostasis and survive in bio lms under oxygen-depleted environments [3,48].The upregulation of the phenazine biosynthesis genes is further supported by the upregulation in biosynthetic genes (phnAB and pqs operon) of Pseudomonas quinolone signal (PQS), which tightly regulates pyocyanin production.Also, our data is consistent with our prior report that showed that growth in MM9 impacts pyocyanin production and quorum-sensing regulated pathways [14].P. aeruginosa has multiple resistance mechanisms to protect itself from the toxic effects of pyocyanin and maintain redox homeostasis [49].This was observed through the upregulation of expression of genes encoding the monooxygenase PumA and RNA e ux pump MexGHI-OpmG.One operon consisting of two genes associated with cyanide resistance (i.e., PA14_10550 and PA14_10560) was upregulated [50,51].Besides this operon, a thiosulfate sulfur-transferase encoded by PA14_30430 was upregulated.Although this is not experimentally con rmed yet, we hypothesize that the thiosulfate sulfur-transferase (encoded by PA14_30430), which carries repeated rhodanese homology domains, detoxi es cyanide to sul te and thiocyanate.Then, the sul te reductase (encoded by PA14_10550) reduces sul te to sul de.Whether the hypothetical protein encoded by PA14_10560 is possibly involved is yet to be determined.Overall, the results suggest that malonate utilization is tightly linked with anaerobic respiration and associated with the carbon starvation stress response.

Temperature, osmolarity, and pH stress response
Even though all cultures were incubated at 37°C, malonate utilization was associated with heat-shock response on the transcriptomic level (Table 7).This was accompanied by an increase in the expression of four heat-shock genes encoding for proteins (IbpA, HtpX, HtpG, and GrpE) with an average of 5.6 times and decrease in the expression of two cold-shock proteins with an average of 4.6 times.Our results are consistent with the ndings of other groups that indicated heat-shock responses can be induced by conditions that did not alter temperature such as alkaline pH or carbon starvation [52][53][54].
Finally, we observed the upregulation of certain genes involved in osmolarity and pH uctuations (Table 7).For example, the expression of the transcriptional regulator OmpR was signi cantly upregulated.Among its regulated genes are htpX (membrane protease), PA14_72930 (predicted lipidbinding transport protein, Tim44 family), and PA14_30410 (YccA-like protein).All of ompR, htpX, PA14_72930, and PA14_30410 protect P. aeruginosa against osmolarity and pH stressors [55].The role of these genes involves maintaining membrane integrity.Because of this critical role, the function of those genes involves the protection against aminoglycoside antibiotics [55].Overall, these results suggest that malonate utilization induces a global stress response in P. aeruginosa that spans various physical and physiological stressors.However, it is not clear what these adaptations are responsive to exactly, which we aimed to address next.

Gene expression in CF isolates
After observing that many of the clinical isolates show similar phenotypes to that of PA14, we wondered if we could also observe similar trends at the transcriptional level.To evaluate this, we selected ve clinical strains that showed variable growth phenotypes in MM9 and GM9 and examined their expression of stress response genes.Brie y, MUC 16 was selected as it showed no signi cant growth difference in MM9 and GM9, MDR 2588 and MUC 148 showed higher growth in MM9, and MUC 135 and MUC 100 showed higher growth in GM9.We examined the expression of genes related to stress response that we identi ed to be upregulated in MM9.These included genes belonging to metal, oxidative, anaerobic, and temperature stress responses.While we observed a signi cant variability in the gene expression of the selected genes, a few showed similar trends (Fig. 2D).Speci cally, genes involved in metal and oxidative stress response (PA14_53300 and PA14_18070) were consistently upregulated in MM9 vs. GM9 in four out of the ve selected clinical isolates, regardless of their growth preference (Fig. 2D), like what was previously observed in PA14.In general, malonate utilization appears to be linked to comparable stress response gene expression patterns and phenotypes among numerous clinical isolates and laboratory strains of P. aeruginosa.

Aluminum is accumulated intracellularly in the presence of malonate
Because the transcriptomic analysis revealed a striking metal stress response, we hypothesized that malonate utilization results in the accumulation of metal ions within the bacterial cells.To determine the effect of malonate or glycerol as sole carbon sources on the accumulation of various metal ions within P. aeruginosa cells, we used inductively coupled plasma mass spectrometry (ICP-MS) to detect the level of metal ions.Because chemicals may carry metal impurities, we used Chelex-treated M9 and Chelextreated carbon sources.Chelex is a chelating agent that binds polyvalent metal ions; thus, this treatment removed the possibility that metal changes in the different treatments could be driven by trace metal contaminates in the different carbon sources.This procedure eliminated several metals essential for growth.Therefore, to complement those metals, we added a cocktail of these essential metal ions to the prepared media (i.e., Mg 1mM, Mn 25 µM, Ca 100 µM, Fe 5 µM, Zn 25 µM, Co 0.1 µM, and Cu 0.1 µM).After overnight incubation of P. aeruginosa in Chelex-treated GM9 and MM9, cells were collected and then prepared using an established protocol following slight modi cations [56].Using ICP-MS analysis of the overnight cultures, we measured the concentration of 17 metal ions (Fig. 4).While most of the measured metal ions were similarly abundant in cells obtained from GM9 and MM9, two metal ions (i.e., Al and Sr) showed a statistically signi cant difference between MM9 and GM9.Speci cally, Al and Sr were ~ 44 and ~ 9 times, respectively, more abundant intracellularly in MM9 versus GM9 (Fig. 4).The mechanism by which malonate utilization induces the accumulation of metal ions is not clear yet.One possible explanation might be attributed to the lower level of pyoverdine in MM9 since this molecule can decrease toxic metal accumulation [57,58].Thus, a decrease in pyoverdine production might have enabled this metal accumulation in MM9.
Because we observed an upregulation in all known regulons related to copper stress response, we expected to nd Cu to be accumulated in MM9.Surprisingly, this was not the case (Fig. 4).One possible explanation is that while Cu gets accumulated at rst, P. aeruginosa then adapts to get rid of excess Cu through the production of a secreted copper-containing small molecule named uopsin C [59].This hypothesis is strengthened by the prominent upregulation (over 100 times higher in MM9 vs. GM9) of the biosynthetic operon consisting of ve genes PA14_18810-18860 that is responsible for uopsin C biosynthesis.
Motivated by the phenotype of P. aeruginosa, in which its malonate utilization induces the expression of biosynthetic genes of uopsin C and pyocyanin, and knowing both of these molecules act as antibiotics against Staphylococcus aureus [59, 60], we hypothesized that this could bene t P. aeruginosa in its competition against other bacteria it may encounter in the lung environment.We further hypothesized that the survival of S. aureus will decrease when co-cultured with P. aeruginosa in MM9, but not in GM9.To test this hypothesis, we used S. aureus strain JE2, an established methicillin-resistant lab strain [61], to study its survival in the presence of P. aeruginosa in MM9 and GM9.A co-culture of P. aeruginosa and S. aureus in a 1:1 ratio was grown in both MM9 and GM9 overnight.Then, cells were serially diluted and plated on selective media.First, we con rmed that no difference was observed in the growth of P. aeruginosa when co-cultured with S. aureus (Supplementary Fig. 3A).In contrast, we observed a signi cant reduction in the number of S. aureus cells when co-cultured with P. aeruginosa in MM9, while no such difference was observed when co-cultured with P. aeruginosa in GM9 (Supplementary Fig. 3B).While the higher levels of pyocyanin and possibly uopsin C in MM9 as compared to GM9 may explain the increased death rate of S. aureus in MM9, other virulence or metabolic factors may contribute to this phenotype as well.
Genetic requirements for P. aeruginosa to grow using malonate as a sole carbon source The observed upregulation in stress-related genes raises an interesting question: Are these stressrelated genes essential for the growth of P. aeruginosa in MM9 to cope with the stress induced by malonate utilization?To address this question, we performed a high-throughput screening on a commercially available transposon mutant library of P. aeruginosa UCBPP-PA14 containing over 5,500 unique mutants [62].We grew wild-type PA14 and the mutants in GM9 and MM9 and measured bacterial growth using optical density (OD 600 ).We observed a variable number of preliminary candidate mutants showing no growth in either MM9, GM9, or both (Supplementary Table 1).Upon the completion of the primary screening, we selected the hits of interest that overlapped with our transcriptomics dataset for further testing.We selected these mutants along with the wild-type (WT) PA14 to reassess their growth in MM9 and GM9 (Supplementary Fig. 4A and 4B, Fig. 5).While some mutants showed a statistically signi cant difference in their growth in comparison to that of WT, only ∆PA14_27480 showed defective growth in both MM9 and GM9.The expression of PA14_27480 was upregulated 8 times in MM9 vs. GM9 (Table 7).PA14_27480 codes for the heat shock protein HtpX.HtpX and other heat-shock proteases allow P. aeruginosa to cope with protein misfolding stress induced by high temperature, carbon starvation, or alkaline pH, thus promoting its survival [54].Eight mutants showed differential growth in MM9 or GM9, four of which showed more defective growth in MM9 rather than GM9 (Fig. 5).∆PA14_18850 caught our attention because its expression was ~ 150 times higher in MM9 vs. GM9.Furthermore, PA14_18850 codes for FlcB, which catalyzes the rst step in uopsin C biosynthesis.Because uopsin C plays a role in copper detoxi cation, this suggests that this functional adaptation is important for P. aeruginosa to cope with metal stress response in MM9.

Discussion
Bacteria occupying different niches is often dependent on their carbon-source utilization versatility.This can in turn have a profound impact on their growth rate, virulence, phenotypes, and resistance to antibiotics [9,[63][64][65][66].Earlier studies have described the role of carbon sources in the metabolism of P. aeruginosa [67][68][69][70].Our study highlights the role of malonate as a sole carbon source in inducing a global stress response in P. aeruginosa.
We rst asked if phenotypes associated with malonate-utilization were similar in lab and clinical strains of P. aeruginosa.Interestingly, most of the tested strains demonstrated better growth in MM9 vs. GM9.This result is of high clinical relevance because the traditional lab medium for selective growth of P. aeruginosa is Pseudomonas Isolation Agar (PIA), which requires glycerol to be added to it (2%) to serve as an energy source and to enhance pyocyanin production (BD Diagnostics or Remel).If strains of P. aeruginosa do not utilize glycerol as a carbon source, this can hinder isolating such strains that may utilize another carbon source such as malonate or mischaracterize P. aeruginosa as malonate is a much better enhancer of pyocyanin production than glycerol [14].This is further supported by previous studies that investigated nutrient availability in sputum samples.For example, a study showed that malonate is among the abundant metabolites in the sputum of healthy volunteers [17].However, the abundance of malonate in the sputum of cystic brosis patients is an important issue that warrants further investigation.Therefore, our results highlight the importance of studying the nutrient adaptation of different strains of P. aeruginosa to assist in their identi cation and isolation.Furthermore, we showed that the expression of certain genes involved in stress response are similarly overexpressed in clinical strains of P. aeruginosa.Overall, our ndings show that clinical strains have unique transcriptional features that may deeply impact P. aeruginosa's adaptation to speci c environments found inside the host.Further genomic investigations are required on more clinical isolates to decipher potential mechanisms that facilitate their adaptation in response to different carbon sources.Further research on the metabolomics and proteomics analysis of P. aeruginosa exposed to malonate will provide us with valuable insights into the speci c metabolites and proteins that are generated or triggered in the presence of this carbon source.Such pathways can eventually be used as a potential therapeutic target to combat Pseudomonas-associated infections and help us identify new strategies complementary or alternatives to antibiotics to e caciously combat this notorious pathogen.
Our present ndings align with the results obtained in our previous work, demonstrating increased and reduced tolerance to kanamycin and nor oxacin, respectively, in MM9 compared to GM9, for lab strains [18,19].In this latest investigation, we increased the spectrum of our lab strains and incorporated clinical strains to reinforce and validate our earlier observations.Most clinical strains, including the three lab control strains, exhibited heightened tolerance to kanamycin in MM9 compared to GM9.Conversely, all clinical strains and the three lab strains, demonstrated increased susceptibility to nor oxacin in MM9 as opposed to GM9.
Next, we wanted to investigate the transcriptional changes associated with this carbon source.It is known that the differential activation of the glyoxylate cycle in the presence of different carbon sources can affect both the pathogenesis and virulence of P. aeruginosa [71].Our transcriptomic data pointed out that, in the presence of malonate, P. aeruginosa implements speci c pathways to survive and adapt to its environment, modifying its virulence and stress response.In our study, we found that both the glyoxylate and methyl citrate cycle-associated genes were upregulated in the presence of malonate.The transcriptomic data further highlighted the intimate interaction between malonate utilization and various stress responsive genes in P. aeruginosa.Our transcriptomic data showed an upregulation in genes associated with metal stress response.This intrigued us to examine the metal ions accumulation in the presence of malonate.Aluminum and strontium were the two main metals that showed a signi cant accumulation in MM9 vs. GM9.We speculate that aluminum accumulation could be the result of differential siderophore production [72].While we expected to observe copper accumulation in MM9, we observed none.We speculate that as an adaptation strategy, P. aeruginosa eliminates excess copper through the production and secretion of the copper-containing small molecule uopsin C [59].This is supported by the remarkable upregulation of the uopsin C-biosynthetic genes.
Because P. aeruginosa and S. aureus are known to cause chronic infections, including lung infections, we were curious if malonate in uences their interspecies interaction.A recent study demonstrated that the ability of P. aeruginosa to break down acetoin induces trophic cooperation between S. aureus and P. aeruginosa and improves their survival [73].Our experiments revealed that malonate-utilization allowed P. aeruginosa to outcompete S. aureus.This could be explained by our previous observations showing that malonate induces the expression of biosynthetic genes of uopsin C and pyocyanin, both of which are toxic to S. aureus [59,60,74].We excluded the possibility that acetoin could be involved here because our transcriptomic data showed an upregulation in metabolic genes of acetoin, which should be improving S. aureus tness, but was not observed here.However, other virulence factors not examined in our study could be involved too.This suggests that in a niche where malonate is available as a carbon source, it can allow P. aeruginosa to outcompete S. aureus.
Overall, we present malonate as an important carbon source to P. aeruginosa that can in uence its carbon metabolism, elicit global stress response, and lead to intracellular metal accumulation, improving its competitiveness against other microbes.Furthermore, phenotypes associated with malonateutilization are not speci c to lab strains of P. aeruginosa, but also to clinical strains that warrant further investigation.

Bacterial growth and strains
Bacterial strains used in this study are described in Tables 2 and 3. We used the strain PA14 and its isogenic mariner transposon mutants (Supplementary Table for most of the experiments.Bacterial strains were routinely grown overnight in Luria-Bertani (LB) broth.When needed, antibiotics were added at the following concentrations: 50 µg/mL of ampicillin and 15 µg/mL of gentamicin.
For analysis of the effect of malonate as a sole carbon source on the growth and virulence of PA14, we used the established M9 minimal medium (6.0 g Na 2 HPO 4 , 3.0 g KH 2 PO 4 , 0.5 g NaCl, 1.0 g NH 4 Cl per L supplemented with 0.2 mM CaCl 2 and 2 mM MgSO 4 ) (Fisher Scienti c) as a basal medium [75].No iron source was added to the M9 minimal medium.For the control medium, we modi ed the M9 by adding 1% glycerol (v/v; 110 mM) as a sole carbon source (GM9).We previously utilized this concentration of glycerol as a carbon source in analyzing the regulation of P. aeruginosa virulence genes [14].For M9 media containing malonate as a sole carbon source (MM9), we added 40 mM malonate (or 100 mM malonate if explicitly mentioned), as sodium malonate dibasic (MilliporeSigma, St. Louis, MO).For analysis of gene expression and virulence factor production, PA14 was routinely grown in GM9 and MM9 for 16 hours.

RNA extraction
PA14 in MM9 and GM9 media was grown overnight for 16 hours at 37°C in shaking condition.The overnight grown culture was then centrifuged at 5,000 rpm speed for 10 min.After discarding the supernatant, bacterial pellets were lysed by the addition of lysozyme and proteinase K for 15 min at room temperature.RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol.RNA solution was digested with the RNase-free DNase set (Qiagen), followed by on-column DNase digestion to eliminate any remaining traces of genomic DNA.The puri ed RNA was quanti ed using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE).The samples were then sent to Genewiz for library prep and Illumina HiSeq.Only samples with an RNA integration number greater than 8.0 were used for cDNA library preparation.
Transcriptomic (RNA-seq) analysis RNA-seq data were analyzed using Rockhopper software implementing reference-based transcript assembly with UCBPP-PA14 as a reference genome followed by calculating the fold change for the transcripts at each growth condition [20].NCBI Reference Sequence NC_008463.1 was used.Datasets were normalized using upper quartile normalization, then transcript abundance was quanti ed using reads assigned per the kilobase of target per million mapped reads normalization method.The selection for differential expression required genes to have a fold change of ≥ 1.5 and a Q value of ≤ 0.05 to be considered signi cant.The Q value was obtained by adjusting the P value using the Benjamini-Hochberg procedure.
Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) One µL of RNA was used for cDNA synthesis.For qRT-PCR, equal amounts of cDNA were mixed with iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) together with 2 µM of speci c primers for each gene examined.Ampli cation and detection were performed using CFX96 Deep Well Real-Time PCR System (Bio-Rad) and analysis of gene expression was done using CFX Manager 3.1 software (Bio-Rad).Each experiment consisted of three biological replicates analyzed in triplicate.Quantity of cDNA in the samples was normalized using the 16S ribosomal RNA gene PA14_08570.A list of primers used in this study are mentioned in Supplementary Table 2. Bacterial cells were cultured overnight in Chelex-treated MM9 and GM9 for 16 hours in the presence of a cocktail of essential metal ions add to the media (Mg 1mM, Mn 25 µM, Ca 100 µM, Fe 5 µM, Zn 25 µM, Co 0.1 µM, and Cu 0.1 µM).These cultures were pelleted and washed in Chelex-treated 500 µL phosphate-buffered saline (PBS).Next, the pellet was suspended in 1 mL metal-free water (Chelex treated) and sonicated at 100% amplitude for 30 seconds each.The probe was washed with Chelextreated water and the control solution used was sonicated water.The probe was wiped and rinsed between samples as thoroughly as possible using water fresh from our ltration system.We sonicated 1 water sample between every 2 samples to account for metal uctuations that may occur over the sonication process.Samples were normalized to equal protein concentration and 100 µL of each sample was digested by adding 1 mL 50% HNO3 (Optima grade; Fisher) followed by overnight incubation at 50°C in a metal-free 15-ml conical tube.After digestion, samples were diluted to a 20-ml nal volume in Milli-Q water and submitted for inductively coupled plasma mass spectrometry (ICP-MS) analysis.Levels of Ag, Al, As, B, Be, Cd, Co, Cr, Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Ti, U, V, and Zn were measured.Quality analysis and quality control included Rh-103 and In-115 as internal standards as well as blanks and continuing calibration checks which were run every 10 samples.Calibration standards were prepared in 50 mL polypropylene tubes with 2% HNO 3 to match the sample matrix.Detection limits for the elements quanti ed were Li = Cu = 0.01 ppb, Be = 0.0006 ppb, Mg = Al = 0.47 ppb, V = 0.04 ppb, Cr = Mn = Cs = Rb = U = 0.001 ppb, Fe = 0.04 ppb, Co = 0.005 ppb, Ni = Sr = 0.02 ppb, Ga = 0.002, Zn = 0.11 ppb).Ion levels were normalized to the endogenous ion levels of the control samples.

Co-culture in MM9 and GM9 media
Prior to inoculation of cultures into MM9 and GM9 growth medium, the laboratory reference strain of P. aeruginosa UCBPP-PA14 was cultured in LB and the wild-type strain of S. aureus, USA300 JE2 was cultured in Tryptic soy broth (TSB).Overnight cultures were washed thrice with lter-sterilized PBS to remove media contamination from either LB or TSB.After a nal wash with M9, cells were normalized to an OD 600 of 1.0 (culture density of ~ 10 8 ).Normalized cells were then suspended in MM9 and GM9 as a monoculture or co-culture.Cells were then incubated as monocultures or co-cultures for 24 hours at 37°C under shaking condition.Following incubation, bacterial cells were diluted in sterile PBS and plated on selective media, P. aeruginosa monocultures were plated on Pseudomonas Isolation Agar (PIA) plates, S. aureus monocultures were plated on Mannitol Salt Agar (MSA) plates, and co-cultures were plated on both plates to observe differences in microbial growth.
Antibiotic susceptibility testing for bacterial strains inhibitory concentration (MICs) of kanamycin and nor oxacin against and clinical isolates of P. aeruginosa was determined in both MM9 and GM9 using broth microdilution method [77].
Overnight cultures were adjusted to an optical density at 600 nm of 1.0 in 1 M9 and diluted to ~ 5 10 5 colony-forming units (CFU) per ml.Both kanamycin and nor oxacin were diluted in 1 M9, then added to 96-well plates containing bacterial inoculum in either MM9 or GM9.Cells without any drug were also incubated as a growth control.Distilled water and glacial acetic acid were added as vehicle controls for kanamycin and nor oxacin, respectively, as the initial drug stocks were prepared.Plates were incubated at 37°C for 24 hours at static condition.Bacterial growth was assessed using 20µL of 0.2 mg/mL resazurin solutions in each well.Following another 12 hours of incubation with resazurin, the MIC was determined visually by observing the transition from blue (non-viable) to pink (viable) color.We standardized the MIC data in MM9 against GM9 and represented the results in a heatmap for comparative analysis.A value of 1, denoted by a yellow color in the heatmap, indicated that a strain remained unaffected by the action of any tested antibiotic in both media.Increasing tolerance to a speci c antibiotic in MM9 was represented by shades of light to deep green, while greater susceptibility was depicted by shades of light to deep purple.

Statistical Analysis
Statistical analyses and graphics plotting were performed using R 4.0.5 and GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA).Unpaired t-test was used to determine statistical signi cance (unless otherwise stated).Declarations     Effect of malonate utilization on accumulation of metal ions in P. aeruginosa.(A) Accumulation level of all measured metal ions using ICP-MS.PA14 was grown in Chelex-treated malonate and glycerol supplemented Chelex-treated M9.Samples were processed for ICP-MS analysis as mentioned in the Experimental Procedures.Samples were normalized based on protein content, then analyzed for the accumulation of the metal ions using ICP-MS.Error bars represent 1 standard deviation of the results from triplicate samples.Unpaired t-test (two-tailed) was used to measure statistical signi cance.* P < 0.05 and ** P < 0.01.The concentration of metal ions was measured in parts per billion (ppb).This experiment was repeated three times on three different days and repeated once using the previously used carbon source concentrations and once using equimolar concentrations.
aggregation index was calculated as the ratio of post-sonication OD 600 to pre-sonication OD 600 of overnight cultures[76].Intracellular metal ions measurement using ICP-MSSamples were prepared for ICP-MS following an established protocol with slight modi cations[56].

Figures Figure 1
Figures

Table 2
Description of lab strains used in this study.

Table 3
MIC of Kanamycin (KAN) and nor oxacin (NOR) against wild-type and clinical isolates of P. aeruginosa in MM9 and GM9 media.ng: no growth, ng-aa: no growth with glacial acetic acid as vehicle control.

Table 4
Differentially expressed genes in PA14 grown in MM9 in comparison with GM9.Listed genes are related to metal stress response.

Table 7
Differentially expressed genes in PA14 grown in MM9 in comparison with GM9.Listed genes are related to temperature, osmolarity, and pH stress responses.